Kinesin motor modulators derived from the marine sponge Adocia

ABSTRACT

This invention provides novel compounds derived from a marine sponge, Adocia sp., that specifically modulat kinesin activity by targeting the kinesin motor domain and mimicking the activity a microtubule. The compounds act as potent anti-mitogens are useful in a wide variety of in vitro and in vivo applications.

CROSS-REFERENCE TO RELATED INVENTIONS

[0001] This is a continuation-in-part of U.S. S No. 60/070,772, filed onJan. 8, 1998, which is herein incorporated by reference in its entiretyfor all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Grant No.GM 35252, awarded by the National Institutes of Health. The Governmentof the United States of America may have certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0003] Plants and animals have yielded a number of chemical moleculeshaving useful biological activity (e.g., anti-tumor activity).Particularly rich sources of biologically active chemicals are marineorganisms, which comprise over half a million species. Marine organismshave been found to produce a variety of metabolic often havingunprecedented chemical structures.

[0004] In recent years, an increasing number of natural productsextracted from marine organisms have been reported to exhibit a varietyof biological activities such as antimicrobial, antiviral, antifungaland anticancer activities. These include peptides, polyethers,alkaloids, prostanoids, and the like. Such compounds have been obtainedfrom sponges, octocorals, algae, tunicates, nuclibranches, bryozoans andmarine bacteria.

[0005] In particular, a number of anti-tumor and anti-fungal compoundshave been extracted from marine life. For example, U.S. Pat. No.4,729,996 discloses anti-tumor imidazole ring compounds isolated fromthe marine sponges Teichaxinella morchella and Ptioocaulis walpersi.U.S. Pat. No. 4,808,590 discloses nitrogen containing cyclic compoundsisolated having antiviral, anti-tumor, and antifungal properties,isolated from the marine sponge Theoneloa sp. Similarly, U.S. Pat. No.4,866,084 discloses bisindole alkaloids extracted from the marine spongeSpongosorites ruetzleri useful in treating certain classes of tumors,while U.S. Pat. No. 4,970,226 discloses bis-indole imidazole alkaloidsand derivatives isolated from the marine sponge Spongosorites sp. whichexhibit useful anti-tumor and antimicrobial properties.

[0006] Marine sponges, in particular, have proven to be a rich resourcefor biologically active compounds (see, e.g., Scheuer, P. J. (ed.)(1978-1983) Marine Natural Products, Chemical and BiologicalPerspectives Vol. I-V, Academic Press, New York; Faulkner (1977)Tetrahedron, 33: 1421; Faulkner (1984) Nat, Prod. Rep. 1: 551; Faulkner(1986) Nat, Prod. Rep. 3: 1; Faulkner (1987) Nat, Prod. Rep. 4: 539;Faulkner (1988) Nat, Prod. Rep. 5: 613; Faulkner (1990) Nat, Prod. Rep.7: 269; Faulkner (1991) Nat, Prod. Rep. 7: 269; Faulkner (1992) Nat,Prod. Rep. 9: 323; Faulkner (1993) Nat, Prod. Rep. 10: 497; Faulkner(1994) Nat, Prod. Rep. 11: 355; Faulkner (1954) Nat, Prod. Rep. 12: 223;Faulkner (1996) Nat, Prod. Rep. 13: 75; Faulkner (1997) Nat, Prod. Rep.14: 256; and Faulkner (1985) J. Am. Chem. Soc. 107: 4796-4798). Howeverthere exist literally thousands of species of marine sponges and theseorganisms are only beginning to be explored.

SUMMARY OF THE INVENTION

[0007] This invention provides novel compounds derived from a marinesponge, Haliclona (aka Adocia) sp., that specifically modulate (e.g.,inhibit) kinesin activity by targeting the kinesin motor domain andmimicking the activity a microtubule. It is believed this mode ofkinesin motor modulation is previously unknown. Thus, it was also adiscovery of this invention that the kinesin-microtubule interactionsite is a useful target for small molecule modulators of kinesin motoractivity.

[0008] Because the compounds were initially derived from the marinesponge Haliclona (Adocia) sp. they are referred to herein as Adociacompounds or Adocia-derived compounds. Particularly preferredAdocia-derived compounds are adociasulfates. Thus, in one embodiment,this invention provides for the Adocia-derived compounds having theformulas shown herein, more preferably for the adociasulfate compoundshaving the formulas shown herein.

[0009] The Adocia-derived compounds are potent kinesin motor modulatorsthat appear to block kinesin binding of microtubules. The compounds arepotent anti-mitotic agents that are highly effective in vitro and invivo. Thus, in another embodiment, this invention provides compositionfor the in vivo modulation (e.g., inhibition) of kinesin motor activity(e.g., in a cell). The compositions typically comprise any of theAdocia-derived kinesin motor inhibitors described herein in combinationwith a pharmacologically acceptable excipient.

[0010] In another embodiment, this invention provides methods ofmodulating (e.g., inhibiting) kinesin motor activity in a cell. Themethods involve contacting the cell with one or more of theAdocia-derived kinesin modulators described herein. The cell, althoughpreferably a mammalian cell, need not be so limited. Other suitablecells include, but are not limited to, fungal cells and microbial cells.The cell can be in vitro or in vivo. Where the method is practiced in atherapeutic context (e.g., to ameliorate the effects of a pathologicalcondition characterized by hyperproliferation of one or more cells) theAdocia-derived kinesin modulators are preferably administered in atherapeutically effective dose.

[0011] In still another embodiment, this invention provides methods ofassaying a test compound for kinesin modulatory activity. The methodsinvolve contacting a microtubule and a kinesin motor (e.g. a kinesinmotor protein) with one of the Adocia-derived kinesin modulatorsdescribed herein and detecting a change in kinesin motor activityresulting from the contacting. In a particularly preferred embodiment,the method is practiced with one of the kinesin modulators of FormulasI, and III-VI, more preferably with one of the kinesin modulators ofFormulas I or III. The change in motor activity is preferably detectedthrough a motility assay, a binding assay, an ADP release assay, or anassay for anti-mitotic activity. Typically the change in activity isevaluated with reference to a negative control (e.g., typically the sameassay, but lacking a kinesin motor modulator) and/or with reference to apositive control (e.g., typically the same assay, with a differentkinesin motor inhibitor, preferably one whose activity has previouslybeen characterized).

[0012] This invention also provides Adocia-derived kinesin modulatorkits. The kits typically include a container containing one or more ofthe Adocia-derived kinesin modulator described herein. The kits canoptionally include a pharmacological excipient and/or a deliveryvehicle. When the excipient and/or delivery vehicle are provided theymay be provided combined with the kinesin motor inhibitor or in aseparate container for combination at the time of use. The kit can alsoinclude instructional materials describing the use of the compounds inany of the methods described herein.

[0013] In still another embodiment, this invention provides methods ofmodulating kinesin motor activity. The methods involve contacting thekinesin motor with a small organic molecule that competitively inhibitsthe kinesin motor at a microtubule binding site. In a particularlypreferred embodiment, the small organic molecule is an Adocia-derivedkinesin modulator as described herein or a a small organic molecule isidentified according to the methods described herein.

[0014] This invention also provides methods of identifying an agent thatmodulates the kinesin inhibitory activity of an Adocia kinesin inhibitor(e.g., on of the Adocia sulfates or Adocia derived kinesin inhibitorsdescribed herein). The methods involve contacting a microtubule and/or akinesin motor and/or an Adocia kinesin inhibitor with a candidate agent;and detecting a change in the kinesin inhibitory activity of the Adociakinesin inhibitor resulting from the contacting, wherein a changeindicates the identification of an agent that modulates the kinesininhibitory activity of the Adocia kinesin inhibitor.

[0015] Methods are also provided for identifying an agent thatinterferes with the binding of an Adocia kinesin inhibitor with akinesin. These methods involve contacting a kinesin and an Adociakinesin inhibitor (e.g. an adocia sulfate or an adocia derived kinesinmodulator described herein) with a candidate agent; and detecting adecrease in the binding of the Adocia kinesin inhibitor with the kinesinresulting from said contacting, wherein a decrease indicates theidentification of an agent that interferes with the binding of theAdocia kinesin inhibitor and the kinesin.

[0016] Also provided herein is a complex comprising an Adocia kinesininhibitor and a kinesin.

[0017] Methods are also provided for modulating cellular growth in anorganism (e.g., an animal or a plant). The methods preferably involveadministering to the organism a composition comprising apharmaceutically acceptable carrier any one or more of the compoundsdescribed herein (e.g. adocia sulfates or adocia-derived kinesinmodulators) in a quantity sufficient to alter said cellular growth in anorganism

[0018] Definitions

[0019] The term “molecular motor” refers to cytoskeletal molecule(s)that utilize chemical energy to produce mechanical force, and drive themotile properties of the cytoskeleton.

[0020] The terms “kinesin” and “kinesin superfamily” as used hereinrefer to a superfamily of eucaryotic motor proteins used to transport alarge variety of cargoes along microtubule “tracks”. Members of thekinesin superfamily are believed to be essential for mitotic and meioticspindle organization, chromosome segregation, organelle and vesicletransport and many other processes that require microtubule basedtransport. The common feature of kinesins in the presence of a conserved˜350 amino acid motor domain which harbors the microtubule binding,ATP-hydrolyzing, and force transducing activities (see, e.g. Barton etal. (1996) Proc. Natl. Acad. Sci. USA, 93(5): 1735-1742, and Goldstein,(1993) Annu. Rev. Genet., 27: 319-351).

[0021] The term “kinesin motor” is used to refer to one or more proteinsinvolved in the transduction of chemical energy into mechanical energy.Kinesin is a force generating enzyme that hydrolyzes ATP to ADP andP_(i) and uses the derived chemical energy to induce plus end directedmovement along microtubules. This ubiquitous microtubule motor isthought to power anterograde organelle transport along microtubules. Theterm kinesin motor is intended to include kinesin related proteinsinhibition of which inhibits kinesin motor activity. Kinesin heavy andlight chains have been cloned and sequenced from a number of speciesincluding, but not limited to Drosophila (GenBank M24441), squid opticlobe (GenBank J05258), sea urchin and human (GenBank X65873), and rat(M75146, M75147, M75148), and the like (see, e.g., Yang et al. (1989)Cell 56: 879-889, Wright et al. (1991) J. Cell. Biol., 113: 817-833,Navone et al. (1992) J. Cell. Biol., 117: 1263-1275, and Cyr et al.(1991) Proc. Natl. Acad. Sci. USA, 88: 10114-10118). In addition, thescientific literature is replete with detailed descriptions of kinesins(kinesin motors) and kinesin related proteins (see, e.g., Kreis and Vale(1993) Guidebook to the Cytoskeletal and Motor Proteins, OxfordUniversity Press, Oxford, Vale (1990) Curr. Opin. Cell. Biol, 2: 15-22;Vale (1987) Ann. rev. Cell. Biol., 3: 347-378; and references therein).

[0022] The terms “kinesin motor inhibitor” or “inhibition of kinesinmotor activity” refers to the decrease or elimination ofkinesin/microtubule mediated transduction of chemical energy (e.g. asstored in ATP) into mechanical energy (e.g., force generation ormovement). Such a decrease can be measured directly, e.g., as in amotility assay, or alternatively can be ascertained by the use ofsurrogate markers such as a decrease in the ATPase activity of thekinesin protein, and/or a decrease in the affinity and/or specificity ofkinesin motor protein-microtubule binding interactions, and/or in adecrease in mitotic activity of a cell or cells. Conversely, a “kinesinmotor agonist” or “upregulator of kinesin motor activity” refers to theincrease of kinesin/microtubule mediated transduction of chemical energy(e.g. as stored in ATP) into mechanical energy (e.g., force generationor movement).

[0023] An “Adocia-derived compound” or “Adocia-derived kinesinmodulator” as used herein refers to any of the kinesin modulatorsdescribed herein (see, e.g. Formulas I, III, IV, V, and VI). It will beappreciated, that while the Adocia-derived compounds include naturalproducts derived from sponges (or other marine organisms) the term alsocontemplates analogues of such compounds as described herein. TheAdocia-derived compounds thus need not exist as natural products and maybe chemically synthesized de novo.

[0024] The term “test compound” refers to a compound whose anti-kinesinmotor activity it is desired to determine. Such test compounds mayinclude virtually any molecule or mixture of molecules, alone or in asuitable carrier.

[0025] The term “detecting the binding” means assessing the amount of agiven second component that binds to a given first component in thepresence and absence of a test composition. This process generallyinvolves the ability to assess the amount of the second componentassociated with a known fixed amount of the first component at selectedintervals after contacting the first and second components. This may beaccomplished e.g., by attaching to the second component a molecule orfunctional group that can be visualized or measured (e.g., a fluorescentmoiety, a radioactive atom, a biotin that can be detected using labeledavidin) or by using ligands that specifically bind to the secondcomponent. The level of binding is preferably detected quantitatively.Binding, or a change in binding is indicated at the first detectablelevel. A change in binding, which can be an increase or a decrease, orpresence versus absence, is preferably a change of at least about 10%,more preferably by at least about 20%, still more preferably by at leastabout 50%, still even more preferably by at least about 75%, even morepreferably by at least about 150% or 200% and most preferably is achange of at least about 2 to about 10 fold (e.g., as compared to acontrol).

[0026] The phrase “detecting a change in the kinesin inhibitory activityof he Adocia kinesin inhibitor resulting from said contacting” refers todetermining the presence or absence or quantifying the alteration inkinesin inhibitory activity caused by a particular candidate agentassays for such determinations are further described herein. A change inactivity, which can be an increase or a decrease, or presence versusabsence, is preferably a change of at least about 10%, more preferablyby at least about 20%, still more preferably by at least about 50%,still even more preferably by at least about 75%, even more preferablyby at least about 150% or 200% and most preferably is a change of atleast about 2 to about 10 fold (e.g., as compared to a control).

[0027] The phrase “detecting a change in kinesin motor activityresulting from said contacting” refers to determining the presence,absence or quantifying the alteration in kinesin motor activity causedby a particular composition (e.g., a test compound). The detecting caninvolve any one or more of a variety of assays for kinesin motoractivity as described herein. A change in activity, which can be anincrease or a decrease, or presence versus absence, is preferably achange of at least about 10%, more preferably by at least about 20%,still more preferably by at least about 50%, still even more preferablyby at least about 75%, even more preferably by at least about 150% or200% and most preferably is a change of at least about 2 to about 10fold (e.g., as compared to a control).

[0028] The term “compound” as used herein refers to organic or inorganicmolecules. The term includes, but is not limited to polypeptides,proteins, glycoproteins (e.g. antibodies), nucleic acids,oligonucleotides, and inorganic molecules.

[0029] The term “small organic molecule”, as used herein, refers to acompound that is an organic molecules of a size comparable to thoseorganic molecules generally used in pharmaceuticals. The term excludesbiological macromolecules (e.g., proteins, nucleic acids, etc.).Preferred small organic molecules range in size up to about 5000 Da,more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0030] A “bioagricultural compound” as used herein refers to a chemicalor to a biological compound that has utility in agriculture or inenvironmental and functions to foster food or fiber crop or cropprotection or yield improvement. For example, one such compound mayserve as a herbicide to selectively control weeds, as a fungicide tocontrol the spreading of plant diseases, as n insecticide to ward offand/or destroy insect, mite, and other arthropod pests. In addition, onesuch compound may demonstrate utility in seed treatment to improve thegrowth environment of a germinating seed, seedling, ro young plant as aplant regulator or activator. Other compounds can serve in environmentalmanagement such as, for example, forest management.

[0031] By “protein” herein is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides, andpeptides. The protein may be made of naturally occurring amino acids andpeptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline, and norleucine are considered amino acids for the purposesof this invention. “Amino acid” also includes imino acid residues suchas proline and hydroxyproline. The side chains may be in either the (R)or the (S) configuration. In the preferred embodiment, the amino acidsare in the (S) or L-configuration. If non-naturally occurring sidechains are used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations.

[0032] The terms “nucleic acid” or “oligonucleotide” or grammaticalequivalents herein refer to at least two nucleotides covalently linkedtogether. A nucleic acid of the present invention is preferablysingle-stranded or double stranded and will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage et al. (1993) Tetrahedron49(10):1925) and references therein; Letsinger (1970) J. Org. Chem.35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81: 579; Letsinger etal. (1986) Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett.805, Letsinger et al. (1988) J. Am. Chem. Soc. 110: 4470; and Pauwels etal. (1986) Chemica Scripta 26: 141 9), phosphorothioate (Mag et al.(1991) Nucleic Acids Res. 1.9:1437; and U.S. Pat. No. 5,644,048),phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111:2321,0-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc.114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen(1993) Nature, 365: 566; Carlsson et al. (1996) Nature 380: 207). Otheranalog nucleic acids include those with positive backbones (Denpcy etal. (1995) Proc. Natl. Acad. Sci. USA 92: 6097; non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Angew. (1991) Chem. Intl. Ed. English 30: 423; Letsinger etal. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et a. (1994) Nucleoside& Nucleotide 13:1597; Chapters 2 and 3, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook; Mesmaeker et al. (1994), Bioorganic & Medicinal Chem.Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17; TetrahedronLett. 37:743 (1996)) and non-ribose backbones, including those describedin U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASCSymposium Series 580, Carbohydrate Modifications in Antisense Research,Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al. (1995), Chem. Soc. Rev. pp169-176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997page 35. These modifications of the ribose-phosphate backbone may bedone to facilitate the addition of additional moieties such as labels,or to increase the stability and half-life of such molecules inphysiological environments.

[0033] The term “competitive inhibition” is used to refer to competitiveinhibition in accord with the Michaelis-Menton model of enzyme kinetics.Competitive inhibition is recognized experimentally because the percentinhibition at a fixed inhibitor concentration is decreased by increasingthe substrate concentration. At sufficiently high substrateconcentration, V_(max) can essentially be restored even in the presenceof the inhibitor. Conversely, “non-competitive inhibition” refers toinhibition that is not reversed by increasing the substrateconcentration.

[0034] The term “cell” is used to refer to any cell including, but no tlimited to mammalian, fungal, microbial and invertebrate cells.Preferred cells include tumor cells including, but not limited to,carcinomas, including breast, ovary, prostate, skin, and colon; braincancers, including memingioma, glioma, oligodendroglioma, embryoniccancers; sarcomass; leukemias, and lymphomas. Preferred cells alsoinclude neurons. Particularly preferred neurons are those related toneurodegenerative diseases including Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, Frontotemporal Dementias, andArnyotrophic Lateral Sclerosis. Preferred cells further include cellsderived from the gastrointestinal system including esophagus, stomach,intestine, pancreas, liver, lung, heart, and vascular system as sell ascells from the central and peripheral nervous system, kidney, bladder,muscular system and the bone system.

[0035] “In vivo” refers to in the living body of an organism.

[0036] “In vitro” refers to outside the living body, such as, anartificial environment, for example, a test tube or a cell or tissueculture.

[0037] The term “modulate” as used herein refers to increaing ordecreasing an activity of a molecule. Thus, for example, a kinesin motormodulator acts to increase or decrease (inhibit) kinesin motor activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIGS. 1a, 1 b, and 1 c show structures of preferredadociasulfates.

[0039]FIG. 2 shows adociasulfate-mediated inhibition of the microtubulestimulated ATPase of kinesin. The graph shows the dependence of theapparent k_(cat) value of microtubule-stimulated kinesin ATPase in thepresence of varying adociasulfate concentrations.

[0040]FIG. 3 shows that adociasulfate prevents binding of kinesin tomicrotubules. Monomeric kinesin protein K5-351 (3.5 μM), waspreincubated with microtubules (3.6 μM), MgAMP-PNP (2 mM), with orwithout 35 μM adociasulfate (FIG. 1a). After 10 min the mixture wascentrifuged at 80,000×g for 30 min, and supernatants (S) and pellets (P)were analyzed by SDS-PAGE.

[0041]FIGS. 4a and 4 b show that adociasulfate inhibition is competitivewith microtubule, but not with ATP. ATP-concentration dependence wasdetermined by a coupled enzymatic assay with pyruvate kinase and lactatedehydrogenase monitoring changes in absorbance at 340 nm (Huang et al.(1994) J. Biol., Chem., 269: 16508-16511). FIG. 4a shows that theapparent K_(m) for microtubules depends linearly on adociasulfateconcentration. The intercept with the x-axis gives a Ki value of 0.8 μM.b. Apparent Km for ATP does not depend on AS concentration, only V_(max)is affected,

[0042]FIGS. 5a and 5 b show that adociasulfates induce a burst of ADPrelease. To determine the percent of ADP released from kinesin (Hackney(1994) J. Biol. Chem., 269: 16508-16511) 80 μM kinesin was preincubatedwith α-³²P-ATP at room temperature for 15 minutes and then stored onice. 1 μL aliquots of that mixture were diluted into 100 μL of “chasemix” containing 0.5 mg/ml pyruvate kinase, 2 mM phosphoenolpyruvate andvarying concentrations of aldociasulfate (FIG. 1a). At different timepoints, 5 μL aliquots of the chase mix were quenched in 100 μL of 1 MHCl/1 mM ATP/1 mM ADP. The amount of ADP that became accessible topyruvate kinase and was converted to ATP was determined by thin layerchromatography (TLC) on PEI-cellulose followed by phosphoimager(Molecular Dynamics) quantitation. FIG. 4a shows an example of theexperimental trace. FIG. 4b shows that the magnitude of the burst of ADPrelease depends on adociasulfate concentration.

[0043]FIG. 6 illustrates synthetic schemes for the production ofAdocia-derived kinesin inhibitors of this invention.

DETAILED DESCRIPTION

[0044] This invention provides a previously unknown class of specificenzyme modulators that act to modulate binding of kinesin motor proteinsto microtubules and thereby alter kinesin motor activity. It is believedthat prior to this invention, no small organic molecule modulators (thatare not nucleotides or nucleotide analogues) of kinesin motors wereknown or even suspected to exist.

[0045] The kinesin motor modulators of this invention were initiallyderived from the sponge Adocia sp. and thus are referred to herein asAdocia compounds or Adocia-derived kinesin modulators (e.g. kinesininhibitors). Specific preferred Adocia compounds are sulfates andconsequently referred to herein as adociasulfates (AS).

[0046] I. Kinesin Motor Modulators.

[0047] A) Uses of Kinesin Motor Modulators.

[0048] The kinesin motor modulators of this invention are useful in awide variety of contexts. In particular, preferred modulators of hisinvention act to inhibit activity of kinesin mediated transport. Thekinesins (members of the kinesin superfamily) are implicated inmicrotubule-mediated transport activities. As such they participate in awide variety of activities including, but not limited to mitotic andmeiotic spindle organization, chromosome segregation, organelle andvesicle transport and many others processes that require microtubulebased transport.

[0049] Modulation (e.g. inhibition) of kinesin motors therefor hasprofound effect on cellular function acting, for example, to inhibitmeiosis and/or mitosis, and consequently inhibiting cellular growthand/or proliferation, e.g. in vitro or in humans and other non-humananimals. As powerful anti-mitotics or anti-meiotics, the kinesininhibitors of this invention have a wide variety of uses, particularlyin the treatment (e.g., amelioration) of, e.g. human and veterinary,pathological conditions characterized by abnormal cell proliferation.Such conditions include, but are not limited to: fungal infections,abnormal stimulation of endothelial cells (e.g., atherosclerosis), solidtumors and tumor metastasis, benign tumors, for example, hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas,vascular malfunctions (e.g., arterio-venous malformations), abnormalwound healing, inflammatory and immune disorders, Bechet's disease, goutor gouty arthritis, abnormal angiogenesis accompanying: rheumatoidarthritis, psoriasis, diabetic retinopathy, and other ocular angiogenicdiseases such as retinopathy of prematurity (retrolental fibroplasic),macular degeneration, corneal overgrowth, corneal graft rejection,neuroscular glaucoma, Oster Webber syndrome, and the like. In addition,it is expected the kinesin motor inhibitors of this invention are usefulin the treatment/mitigation of a number of neurodegenerative disorders.The kinesin motor modulators of this invention also find use in theprevention and treatment of plant diseases caused by, for example, fungi(e.g., Plasmodiophora brassicae (club foot of crucifers), Synchytriumendobioticum (potato black wart disease), Plasmopara viticola (downymildew of grape), Phytophthora infestans (late blight of pato andtomato), etc.), nematodes, insects, mites, or other arthropod pests, orparasitic seed plants (e.g., witchweed (Striga asiatica), dwarfmistletoe (Arceuthobium), etc.). Accordingly, the kinesin motormodulators find use in bioagricultural and environmental managementsettings as herbicides, fungicides, pesticides, or insecticides. In apreferred embodiment, the kinesin motor modulator is administered toplants with a bioagriculturally acceptable carrier or exipient.

[0050] The kinesin motor modulators of this invention also have avariety of in vitro uses as well. For example, they can be used tofreeze cells in a particular stage of the cell cycle for a variety ofpurposes (e.g., in the preparation of samples for of histologicalexamination), in the isolation of nucleic acids from a particular stageof the cell cycle, and so forth.

[0051] The kinesin motor modulators of the invention also find use inthe diagnosis of human and veterinary diseases, conditions, orpathologies associated with abnormal kinesin superfamily function, forexample, disease states or conditions associated with hypersensitivityor resistance to kinesin motor modulators.

[0052] The kinesin motor modulators of this invention show uniquespecificity of kinesin motor/microtubule interactions. They thereforprovide novel lead compounds for the development of highly specificinhibitors or upregulators for kinesin families and subfamilies, thusallowing for precise chemical intervention. In addition, the ability ofthe Adocia compounds of this invention to mimic microtubules in kinesinmotor binding allow the creation of artificial kinesin tracks for use invarious kinesin/microtubule assays (e.g., motility assays). Because oftheir ability to modulate the activity of kinesin motors (the conversionof chemical energy to mechanical activity) the kinesin motor modulatorsof this invention are useful for the production of nano-switches andother nano-devices (e.g., nanometer scale micro machines).

[0053] B) Preferred Kinesin Motor Modulators.

[0054] In a preferred embodiment, this invention provides Adocia-derivedkinesin motor modulators characterized by a compound according toFormula (1)

[0055] in which R¹ and R² are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R¹ and R² isnot H, or R¹ and R² are combined to form a single divalent terpenemoiety selected from the group consisting of hemiterpenes, terpenemonomers and terpene oligomers; X¹ and X² are the same or different andare anionic derivatives of an organic group, an inorganic group or agroup which is a combination of organic and inorganic groups; Y^(+n) isan organic or inorganic cation; m is either 1 or 2; and n is either 1 or2.

[0056] Terpenes are characterized as being made up of units of isoprenein a head-to-tail orientation. The terpenes are further classified bythe number of isoprene units in their carbon skeletons, as shown inTable 1. TABLE 1 Terpene classification Isoprene Units Carbon AtomsClassification 1 5 hemiterpene 2 10 monoterpene 3 15 sesquiterpene 4 20diterpene 5 25 sesterterpene 6 30 triterpene 8 40 tetraterpene >8 >40polyterpene

[0057] As used herein, the general term “terpenoid” encompasseshemiterpenes, terpene monomers, terpene oligomers and meroterpenes.“Terpene monomers” refers to derivatized or underivatized terpenesconsisting of ten carbon atoms. “Terpene oligomers” refers toderivatized or underivatized terpenes having more than ten carbons.“Meroterpenes” refers to terpenes that are directly attached to anyaromatic ring. Terpenoids which are named with a prefix (e.g.,hemiterpenes, triterpenes, etc.) include both derivatized andunderivatized analogs of these terpenoids.

[0058] In a preferred embodiment, the terpendoids are oligomeric. In afurther preferred embodiment, the terpene oligomers are members selectedfrom the group of diterpenes, sesquiterpenes, triterpenes,sesterterpenes and triterpenes. In a still further preferred embodiment,the terpenoid is a triterpene.

[0059] Derivatized terpenes include, for example, terpene alcohols,aldehydes, ketones, ethers and esters. Each of these terms is used inits normal art-accepted manner. In a presently preferred embodiment, theinvention provides compounds which are terpene alcohols. In anotherpresently preferred embodiment, the invention provides compounds whichare terpene ethers. In still a further preferred embodiment, the ethersare cyclic ethers.

[0060] The compounds of the invention can be linear terpenes or terpeneswhich include within their structural framework one or more rings. In apreferred embodiment, the terpenes are polycyclic, preferably havingmore than two rings and more preferably having more than four rings. Aring can be saturated, can contain unsaturation or can be aromatic. Aring can have from four to seven members and can consist of only carbonatoms or carbon atoms in conjunction with heteroatoms. Presentlypreferred heteroatoms include nitrogen, oxygen and sulfur. In apreferred embodiment, the rings are composed entirely of carbon atoms.In another preferred embodiment, a ring contains one or more oxygenheteroatoms. In a still further preferred embodiment, a ring contains asingle oxygen atom.

[0061] A compound of the invention can have either one or two terpenoidsattached to the benzene nucleus. When two terpenoids are present theycan be identical or different. Both terpenoids can contain one or morecyclic structures within their framework, both can be linear or one canbe linear and the other can contain one or more cyclic structures withinits framework. In this embodiment, a terpenoid can be attached to thebenzene nucleus as a monovalent moiety or it can be fused to the benzenenucleus as a divalent moiety. In another preferred embodiment, thecompounds consist of one terpenoid attached to the benzene nucleus. In afurther preferred embodiment, the terpenoid has at least one cyclicstructure within its framework. In a preferred embodiment, the terpenoidis fused to the benzene nucleus.

[0062] The benzene nucleus is functionalized with two anionic groups.The anionic groups can be either the same or different and they arederived from acidic organic groups, acidic inorganic groups or groupswhich contain an acidic inorganic group tethered to an organic group.

[0063] Acidic organic groups are primarily derived from carboxylic acidsand thiocarboxylic acids. The carboxylic acids are attached to thebenzene ring directly or through a hydrocarbon chain of between one andfive carbon atoms. In preferred embodiments, the organic acid isattached directly to the benzene nucleus (e.g., phenylformic orphenylthioformic acid) or through a one carbon spacer (e.g.,phenylacetic or phenylthioacetic acid).

[0064] When a hydrocarbon chain is present, this chain can besubstituted with groups which have the effect of modulating the acidityof the acid. Thus, electronegative groups (e.g., F, Cl, Br, NO₂, etc.)attached to the hydrocarbon chain increase the acidity of the attachedacidic group. In an opposite manner, electropositive groups (e.g.,alkyl, alkenyl) decrease the acidity the hydrocarbon chain can beattached to the benzene nucleus through a carbon atom or can be attachedvia a heteroatom such as oxygen (e.g., glycolic acid)

[0065] Inorganic acidic groups are derived from inorganic acidsincluding, but not limited to, phosphoric acid, phosphonic acid,phosphinic acid, boronic acid, sulfuric acid, sulfonic acid, arsonicacid and the like. The acid is bound to the benzene nucleus by eitherthe central atom of the acid, or through an oxygen atom to form an“inorganic ester.” Examples of these two modes of attachment include,for example, phenylphosphinic acid and phenyl phosphate, respectively.In a presently preferred embodiment, the acid is a sulfur containingacid. In an further preferred embodiment the acid is bound to thebenzene nucleus via an oxygen atom. In yet another preferred embodiment,the acid is derived from sulfuric acid.

[0066] Acids which are derived from species consisting of organicradicals and inorganic acids include, for example, alkyl sulfuric acids,alkyl phosphoric acids, alkyl phosphinic acids and the like.

[0067] The cations which are associated with the anionic groups areeither organic or inorganic cations. The cations can have either a ⁺1 or⁺2 charge. When a cation with a ⁺1 charge used, two cations will beassociated with the molecule. When a ⁺2 cation is used, only one cationis necessary.

[0068] Inorganic cations include ions of Groups 1-12. Preferredinorganic cations include, but are not limited to, the cations of Li,Na, K, Cs, Mg, Ca, Mn, Fe, Co, Ni, Cu and Zn. Further preferredinorganic cations are the cations of Li, Na and K.

[0069] Organic cations include, for example, tetraalkyl ammonium salts.The ammonium salts of the present invention are monovalent (e.g.,R₄N⁺Y⁻) or divalent as illustrated by Formula (II).

(R⁴)₃N—(CH₂)_(t)—R³—(CH₂)_(s)—N(R⁴)₃  (II)

[0070] in which R³ is a C₁ to C₁₀ aryl, substituted aryl, alkyl orsubstituted alkyl group and R⁴ is lower alkyl or substituted loweralkyl. The letters s and t represent integers from 1 to 5 and can be thesame or different. When the ammonium salt is monovalent, the alkyl groupthe characteristics of the nitrogen substituents will be generally thesame as those discussed in the context of R⁴.

[0071] A named R group will generally have the structure which isrecognized in the art as corresponding to R groups having that name. Forthe purposes of illustration, representative R groups as enumeratedabove are defined herein. These definitions are intended to supplementand illustrate, not preclude, the definitions known to those of skill inthe art.

[0072] The term “alkyl” is used herein to refer to a branched orunbranched, saturated or unsaturated, monovalent hydrocarbon radicalhaving from 1-10 carbons and preferably, from 1-6 carbons. When thealkyl group has from 1-6 carbon atoms, it is referred to as a “loweralkyl.” Suitable alkyl radicals include, for example, methyl, ethyl,n-propyl, i-propyl, 2-propenyl (or allyl), n-butyl, t-butyl, i-butyl (or2-methylpropyl), etc.

[0073] The term “substituted alkyl” refers to alkyl as just describedincluding one or more functional groups such as lower alkyl, aryl, acyl,halogen (i.e., alkylhalos, e.g., CF₃), hydroxy, amino, alkoxy,alkylamino, acylamino, acyloxy, aryloxy, aryloxyalkyl, mercapto, bothsaturated and unsaturated cyclic hydrocarbons, heterocycles and thelike. These groups may be attached to any carbon of the alkyl moiety.

[0074] The term “aryl” is used herein to refer to an aromaticsubstituent which may be a single aromatic ring or multiple aromaticrings which are fused together, linked covalently, or linked to a commongroup such as a methylene or ethylene moiety. The common linking groupmay also be a carbonyl as in benzophenone. The aromatic ring(s) mayinclude phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenoneamong others.

[0075] “Substituted aryl” refers to aryl as just described including oneor more functional groups such as lower alkyl, acyl, halogen, alkylhalos(e.g., CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy,mercapto and both saturated and unsaturated cyclic hydrocarbons whichare fused to the aromatic ring(s), linked covalently or linked to acommon group such as a methylene or ethylene moiety. The linking groupmay also be a carbonyl such as in cyclohexyl phenyl ketone.

[0076] In a presently preferred embodiment, the organic cation is acompound according to Formula (II). In a further preferred embodiment,R³ is phenyl and R⁴ is lower alkyl. In another preferred embodiment,both R³ and R⁴ are lower alkyl. In these embodiments, s and t arepreferably between 1 and 3, more preferably 1.

[0077] In a further embodiment, the invention provides for compoundshaving a structure according to Formula (III):

[0078] in which R⁵ and R⁶ are either independent monovalent moietiesindependently selected from the group consisting of H, hemiterpenes,terpene monomers and terpene oligomers such that at least on of R⁵ andR⁶ is not H, or R⁵ and R⁶ are combined to form a single divalent moietyselected from the group consisting of hemiterpenes, terpene monomers andterpene oligomers. In a presently preferred embodiment, R⁵ and R⁶ arecombined to form a seven-membered cyclic ether. In another preferredembodiment, R⁵ is a terpenoid and R⁶ is H.

[0079] In another embodiment, the present invention provides foradociasulfate compounds having a structure according to Formula (IV).

[0080] In an additional embodiment, the invention provides anadociasulfate compound having a structure according to Formula (V).

[0081] In a still further embodiment, the present invention provides anadociasulfate compound having a structure according to Formula (VI).

[0082] II. Isolation and/or Synthesis of Adocia-Derived Kinesin MotorModulators.

[0083] The Adocia-derived kinesin motor modulators of this invention canbe created de novo according to standard methods of chemical synthesis.Alternatively, where the modulators are natural products, they can beisolated from the organisms in which they are produced according tostandard methods.

[0084] A) Chemical Synthesis of Adocia Compounds.

[0085] Using the structures provided herein, de novo synthesis of thecompounds of this invention can be achieved using standard methods wellknown to those of ordinary skill in the art. In addition, the compoundsof FIGS. 1a, 1 b, and 1 c, which can be isolated from sponges, asdescribed below, provide convenient substrates which can be modified toproduce the other compounds described herein. De novo synthesis ofterpenoids and/or modification of terpenoids is routine and well knownto those of skill in the art (see, e.g., U.S. Pat. No. 5,596,127 Processfor the continuous preparation of terpene esters; U.S. Pat. No.5,399,724 Acyclic terpene compound; U.S. Pat. No. 5,202,460 Terpenederivatives, their preparation and their use; U.S. Pat. No. 5,073,659Process for the preparation of terpenes; U.S. Pat. No. 4,623,747 Terpenediesters and process for preparing the same; U.S. Pat. No. 4,137,257Terpene hydroxysulfonic acids and corresponding hydroxysulfonate salts;U.S. Pat. No. 4,029,649 Terpene aryl esters, and Ho, (1988) Carbocycleconstruction in terpene synthesis,” New York; Kaufman et al. (1995) J.Med. Chem. 38: 1437-45; Gould, (1995) J. Cell. Biochem. Suppl.22:139-144; and Szirmai et al. (1995) Bioorg. Med. Chem. 3:899-906.Typical synthesis schemes are provide in FIG. 6.

[0086] B) Purification of Adocia Compounds.

[0087] The compounds of the present invention can also be isolated fromnatural sources. The fields of natural products isolation and terpenesynthesis are well developed. Methods of purifying terpenoids of naturalorigin and elucidating their structures are known to those of skill inthe art.

[0088] In a preferred embodiment, the Adocia-derived kinesin motormodulators are purified according to the method described in Example 1.

[0089] III. Assay of Adocia-Derived Kinesin Motor Modulators forActivity.

[0090] It will be appreciated that the different Adocia compounds ofthis invention may exhibit different levels of kinesin motor modulatoryactivity. Consequently, it is desirable to identify those Adocia-derivedkinesin modulators of this invention that exhibit the highest level ofactivity and/or those that show various optimum levels of activity.Thus, in one embodiment, this invention provides methods of assaying(screening) the Adocia-derived kinesin motor modulators for activity.The screening may involve detection of presence or absence of kinesinmotor modulatory activity or quantification of such activity. In apreferred embodiment, such quantification is relative to a controllacking any modulator and/or to a reference kinesin modulator compound.The reference compound may be an modulator of this invention or adifferent modulator. Thus, for example, in a preferred embodiment, thescreened compound will be scored as a strong inhibitor if it hasinhibitory activity equal to or greater than the compounds of FormulasIV, V, or VI, more preferably the compound of formula IV, in a motility,binding, ATPase, or anti-mitotic assay (e.g. the assays described in theExamples herein). Particularly preferred screened compounds exceed theinhibitory activity of the compounds of Formulas IV, V, or VI, mostpreferably Formula IV by a factor of at least 2, more preferably by afactor of at least 5, and most preferably by a factor of at least 10.

[0091] While many assays for kinesin modulation are known to those ofordinary skill in the art, particularly preferred assays includemotility assays, binding assays, and assays for anti-mitotic activity.

[0092] A) Motility Assays.

[0093] Because the microtubule/kinesin motor system transduces chemicalenergy into force generation and molecular movement, motility assaysprovide a convenient means for assaying for modulators of the kinesinmotor proteins; stronger motor modulators producing a greater increaseor decrease in motility or having a particular effect at lowerconcentration. Generally motility assays involve immobilizing onecomponent of the system (e.g, the kinesin motor or the microtubule) andthen detecting movement, or inhibition thereof, of the other component.Thus, for example, in a preferred embodiment, the microtubule will beimmobilized (e.g., attached to a solid substrate) and the movement ofthe kinesin motor molecule(s) will be visually detected. Typically themolecule that is to be detected is labeled (e.g., with a fluorescentlabel) to facilitate detection.

[0094] Methods of performing motility assays are well known to those ofskill in the art (see, e.g., Hall, et. al. (1996), Biophys. J., 71:3467-3476, Turner et al., 1996, Anal. Biochem. 242 (1): 20-5; Gittes etal., 1996, Biophys. J. 70 (1): 418-29; Shirakawa et al., 1995, J. Exp.Biol. 198: 1809-15; Winkelmann et al., 1995, Biophys. J. 68: 2444-53;Winkelmann et al., 1995, Biophys. J. 68: 72S, and the like). Inaddition, a suitable motility assay is described in Example 2.

[0095] B) Binding Assays

[0096] In addition to, or in alternative to, motility assays, bindingassays can also be used to assay (detect and/or quantify) modulation ofkinesin motor proteins. In binding assays, the ability of the putativekinesin motor modulator to inhibit or increase binding of the kinesinmotor protein(s) to microtubules are assayed.

[0097] There are a wide variety of formats for binding assays. In oneembodiment, the microtubule or the motor protein is attached to a solidsupport. The corresponding motor protein or microtubule is thencontacted to the support in the presence of the modulator to be screenedand the amount of bound motor protein or microtubule is then detectedand/or quantified (for suitable binding assay formats, see copendingapplication U.S. S No. 60/057,895, filed on Sep. 4, 1997.

[0098] Solution phase binding assays are also known to those of skill inthe art. For example, in one embodiment, the binding assay is akinesin-microtubule cosedimentation assay (Example 1). In this assay,(pelleting) assay, kinesin binds to microtubules in the presence ofAMP-PNP, a non-hydrolysable analogue of ATP, and sediments to the bottomof the tube after centrifugation to form a pellet. The nonhydrolysableATP analogue permits kinesin-microtubule binding, but not release.Meanwhile, unbound kinesin remains in the supernatant. The negativecontrol (lacking modulator) has saturating amounts of kinesin bound tothe microtubules. In the presence of kinesin motor inhibitors such asthe adociasulfates described herein, most of the motor remains in thesupernatant.

[0099] In one preferred embodiment, the assay, involves

[0100] 1) Adding PEM80 and DMSO (control)/modulator to tube and mixthoroughly;

[0101] 2) Adding other components: Kinesin, microtubules, and 2 mMMgAMP-PNP;

[0102] 3) Centrifuging the tubes (e.g., in Beckman 42.2 rotor at 25,000g, 30 minutes, 20° C.);

[0103] 4. Analyzing the supernatant and pellets on 10% SDS-PAGE.

[0104] Methods of performing kinesin motor-microtubule binding assayscan be found in copending application U.S. S No. 60/057,895 filed onSep. 4, 1997. For a general description of different formats for proteinbinding assays, including competitive binding assays and direct bindingassays, see Stites and A. Terr (1991) Basic and Clinical Immunology, 7thEdition; Maggio (1980) Enzyme Immunoassay, CRC Press, Boca Raton, Fla.;and Tijssen (1985) Practice and Theory of Enzyme Immunoassays, inLaboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, B.V. Amsterdam.

[0105] C) ATPase Assay.

[0106] Kinesin motors are effective ATPases hydrolyzing ATP to ADP toprovide energy for force generation. By examining ADP release fromkinesin in the presence of varying concentrations of kinesin motormodulator (e.g., adociasulfate), the activity of the kinesin motormodulator can be quantified. One such ADP release assay is described inExample 1. In one preferred embodiment, the ATPase activity assayutilizes 0.3 M PCA (perchloric acid) and malachite green reagent (8.27mM sodium molybdate II, 0.33 mM malachite green oxalate, and 0.8 mMTriton X-100). To perform the assay, 10 μL of reaction is quenched in 90μl of cold 0.3 M PCA. Phosphate standards are used so data can beconverted to mM inorganic phosphate released.

[0107] When all reactions and standards have been quenched in PCA, 100μl of malachite green reagent is added to the to relevant wells in e.g.,a microtiter plate. The mixture is developed for 10-15 minutes and theplate is read at an absorbance of 650 nm. If phosphate standards wereused, absorbance readings can be converted to mM Pi and plotted overtime.

[0108] D) Mitotic Activity Assays.

[0109] Assays for mitotic activity typically involve contacting a cell(in vitro or in vivo) the test compound and assaying its effect on theability of the cell proliferate. Alternatively, a mass of cells can becontacted with the compound and the rate of growth of the mass can bemeasured.

[0110] 1) Anti-Mitotic Activity In Situ.

[0111] In one preferred embodiment, anti-mitotic activity of the kinesinmotor modulator of this invention can be assayed in situ by testing theability of the modulator to alter the proliferation of new blood vesselcells (angiogenesis). Such proliferation assays are well known to thoseof skill in the art. One suitable assay is the chick embryochorioallantoic membrane (CAM) assay described by Crum et al. (1985)Science 230:1375. See also, U.S. Pat. No. 5,001,116, which describes theCAM assay.

[0112] Briefly, fertilized chick embryos are removed from their shell onday 3 or 4, and a methylcellulose disc containing a particular amount(e.g., 100 mg) of the compound to be screened is implanted on thechorioallantoic membrane. The embryos are examined 48 hours later and,if a clear avascular zone appears around the methylcellulose disc, thediameter of that zone is measured. Using this assay, a disk of theAdocia-derived compound of this invention is expected to alter cellmitosis and the growth of new blood vessels after 48 hours. Inhibitionof normal blood vessel growth indicates that the Adocia-derived kinesinmodulator is an inhibitor of cell mitosis and angiogenesis.

[0113] 2) Anti-Mitotic Activity In Vitro.

[0114] In vitro assays for anti-mitotic activity are also well known tothose of skill in the art. Typically such assays involve contacting acell (e.g. a cell in culture) with the compound that is to be assayedand determining the effect on cellular proliferation. The cell can beone that proliferates at a normal (e.g. endogenous) rate, or,alternatively, can be a cell in which hyperproliferation has beenstimulated. Measurement of cellular proliferation can be direct (e.g., acell count) or indirect, e.g., through a surrogate marker such as rateof incorporation of a labeled amino acid. Such assays are standard andwell known to those of skill in the art. Descriptions of assays foranti-mitotic activity are found, for example, in U.S. Pat. Nos.5,620,687 and 5,443,962.

[0115] 3) Anti-Mitotic Activity In Vivo.

[0116] In vivo assays for anti-mitotic activity are also well known tothose of skill in the art. Typically these assays involve administeringthe test compound to a subject organism and then evaluating the effectof the compound on a target tissue or organ. Preferred organisms arethose in which a hyperproliferative tissue or organ is manifest. Suchorganisms are well known to those of skill in the art and include, forexample, standard tumor models (e.g., tumors introduced into nude mice)(see, e.g., Sharkey et al. (1990) Cancer Res. 50: 828s-834s). Organismshaving other natural or induced pathological conditions characterized byabnormal cell proliferation are also suitable.

[0117] E) Identifying Specific Modulators of Microtubule Binding.

[0118] As explained above, it was a discovery of this invention thatsmall organic molecules can specifically modulat kinesin activity bytargeting the kinesin motor domain and mimicking the activity amicrotubule. The molecules thus act as competitive inhibitors formicrotubule binding. This is a previously unknown mechanism of kinesin(or other motor, e.g., myosin or dynein) inhibition. Thus, in oneembodiment, this invention provides methods of identifying kinesininhibitors that specifically block the microtubule binding site. It isalso expected that some small organic molecules will facilitateinteractions at the microtubule binding site and similar assays can beused to identify such enhancers of kinesin motor activity.

[0119] Such specific blockers are characterized by the fact that theycan be competitively inhibited by, or competitively inhibit, binders ofthe microtubule binding site, but not binders at the ATPase site. In oneembodiment, this invention therefor provides methods of identifyingcompounds, especially small organic molecules, that change kinesin motoractivity by partially or completely blocking the microtubule bindingsite. The methods involve screening the “test” compound's ability tocompetitively inhibit binding of a moiety (e.g., ATP or an ATP analogue)at the ATPase site and screening the same compound's ability tocompetitively inhibit binding of a moiety (e.g., a microtubule) at themicrotubule binding at the microtubule binding site.

[0120] A kinesin modulator that shows competitive inhibition at themicrotubule binding site, but not at the ATPase site is identified as aninhibitor that specifically binds to the microtubule site.

[0121] Methods of identifying competitive inhibition are well known tothose of skill in the art. Briefly, in the classical Michaelis-Mentonmodel of enzyme kinetics, competitive inhibition is easily recognizedexperimentally because the percent inhibition at a fixed inhibitorconcentration is decreased by increasing the substrate concentration.Thus, where the compound competitively inhibits binding at themicrotubule binding site, increasing the microtubule concentration at afixed concentration of test compound can restore the original(inhibitor-free) maximal rate of reaction (V_(max)). Conversely, wherecompetition is non-competitive increasing the substrate concentrationwill not restore the maximal rate of reaction (Vmax). Assays forspecific inhibition at the microtubule binding site are illustrated inexample 1. For a detailed discussion of analysis of reaction kinetics torecognize competitive, noncompetitive and uncompetitive inhibition, see,e.g., Lehninger (1975) Biochemistry Worth Pub., Inc. New York, N.Y.

[0122] Included herein are assays that identify agents which modulatethe interaction of Adocia kinesin modulators with kinesins. In the casewhere an agent is identified as altering (e.g., interfering with) thebinding of the inhibitor with the kinesin, that agent can be subjectedto further screening to determine its kinesin modulatory activity inaccordance with the assays provided herein. It is understood thatmicrotubules or their components are excluded as interfering agents.Similarly, identified modulators of the inhibitory activity of theAdocia kinesin inhibitors can be subjected to further screening. Inparticular, screens can be performed to determine whether the modulatorswork in conjunction or competitively with the Adocia kinesin inhibitors.Those modulators which function competitively are then further assayedindependently to determine their activity.

[0123] F) High Throughput Screening.

[0124] Conventionally, new chemical entities with useful properties aregenerated by identifying a chemical compound (called a “lead compound”)with some desirable property or activity, creating variants of the leadcompound, and evaluating the property and activity of those variantcompounds. However, the current trend is to shorten the time scale forall aspects of drug discovery. Because of the ability to test largenumbers quickly and efficiently, high throughput screening (HTS) methodsare replacing conventional lead compound identification methods.

[0125] In one preferred embodiment, high throughput screening methodsinvolve providing a library containing a large number of potentialtherapeutic compounds (candidate compounds). Such “combinatorialchemical libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds thus identified can serve as conventional “lead compounds”or can themselves be used as potential or actual therapeutics. In apreferred embodiment, combinatorial chemical libraries are providedcontaining the Adocia-derived compounds described herein.

[0126] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 NPS, 390 NPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).

[0127] A number of well known robotic systems have also been developedfor solution phase chemistries. These systems include automatedworkstations like the automated synthesis apparatus developed by TakedaChemical Industries, LTD. (Osaka, Japan) and many robotic systemsutilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.;Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manualsynthetic operations performed by a chemist. Any of the above devicesare suitable for use with the present invention. The nature andimplementation of modifications to these devices (if any) so that theycan operate as discussed herein will be apparent to persons skilled inthe relevant art. In addition, numerous combinatorial libraries arethemselves commercially available (see, e.g., ComGenex, Princeton, N.J.,Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow,RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md.,etc.).

[0128] Any of the assays for anti-kinesin motor activity describedherein are amenable to high throughput screening. As described above,the adocia-derived compounds are preferably screened for anti-kinesinmotor activity in binding assays, motility assays, or assays foranti-mitotic activity.

[0129] High throughput systems for such screening are well known tothose of skill in the art. Thus, for example, U.S. Pat. No. 5,559,410discloses high throughput screening methods for protein binding, whileU.S. Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methodsof screening for ligand/antibody binding.

[0130] In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configuarablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization. The manufacturers of suchsystems provide detailed protocols the various high throughput. Thus,for example, Zymark Corp. provides technical bulletins describingscreening systems for detecting the modulation of gene transcription,ligand binding, and the like.

[0131] IV. Modulation of Kinesin Motor Activity of Cells.

[0132] In one embodiment, this invention provides for methods ofmodulating kinesin motor activity of cells. It will be appreciated thatwere the cells are cells that hyperproliferate in vivo in any of thepathological conditions described above, the kinesin motor modulators ofthis invention can act as potent anti-mitotic therapeutic agents.However, it will also be appreciated that therapeutic activity is notrequired for all uses of the compounds of this invention. The compoundscan act as significant lead compounds for the development oftherapeutics. Alternatively, the compounds can be used to inhibit growthor proliferation of cells in vitro, to prevent contamination ofbiological samples and the like with pathogenic organisms (e.g., fungi)or to facilitate processing of the biological materials (e.g., inhistological preparations).

[0133] A) Indications.

[0134] As indicated above, the kinesin motor modulators of thisinvention can be used to mitigate a variety of pathological conditions(e.g., in humans and animals) characterized by abnormal cell mitosis.Such diseases include, but are not limited to: abnormal stimulation ofendothelial cells (e.g., atherosclerosis), solid tumors and tumormetastasis, benign tumors, for example, hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas, vascularmalfunctions, abnormal wound healing, inflammatory and immune disorders,Bechet's disease, gout or gouty arthritis, abnormal angiogenesisaccompanying: rheumatoid arthritis, psoriasis, diabetic retinopathy, andother ocular angiogenic diseases such as, macular degeneration, cornealgraft rejection, corneal overgrowth, neuroscular glacoma, Oster Webbersyndrome, and the like.

[0135] The kinesin motor modulators of this invention can also be usedto mitigate variety plant diseases caused by abnormal cell division, orintracellular transport, or parasitic infections caused by, for example,microorganisms, nematodes, insects, and parasitic plants.

[0136] In addition, the kinesin motor modulators can be administered invivo for non-therapeutic purposes (e.g., to rapidly kill and fix cellsfor histological procedures), to elucidate the role of kinesin motors inearly development, and so forth. These applications are not intended tobe limiting, but rather indicative of the multiplicity of different usesfor the kinesin motor modulators of this invention. Other uses of thekinesin motors will be apparent to those of skill in the art.

[0137] B) Compositions for In Vivo Administration.

[0138] The kinesin motor modulators of this invention can beadministered orally, transdermally, by subcutaneous or other (e.g.,intravenous injection, intra-arterial injection, or direct injectioninto the target tissue) injection, intravenously, topically,parenterally, transdermally, or rectally. In addition, the kinesin motormodulators may be incorporated into biodegradable polymers (or otherreservoir) allowing for sustained release, the polymers being implantedin the vicinity of where delivery is desired, for example, at the siteof a tumor. The biodegradable polymers and their use are described indetail in Brem et al., (1991) J. Neurosurg. 74:441-446. The form inwhich the kinesin motor modulator will be administered (e.g., powder,tablet, capsule, solution, emulsion) will depend on the route by whichit is administered: The quantity of the drug to be administered will bedetermined on an individual basis, and will be based at least in part onconsideration of the individual's size, the severity of the symptoms tobe treated and the result sought as described above.

[0139] The kinesin motor modulator compounds are preferably administeredin the form of an acid addition salt thereof, sequentially orsimultaneously with a pharmaceutically-acceptable carrier or diluent,especially and preferably in the form of a pharmaceutical compositionthereof, whether by the topical, oral, rectal, or parenteral (includingsubcutaneous) route, in an effective amount.

[0140] The compositions for administration will commonly comprise asolution of the kinesin motor modulator dissolved or suspended in apharmaceutically acceptable carrier. A variety of carriers can be used,e.g., buffered saline containing suitable emulsifiers, and the like.Methods of producing liposomes and complexing or encapsulating compoundstherein are well known to those of skill in the art (see, e.g., Debs andZhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques6(7): 682-691; Rose U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309;and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414).

[0141] It is recognized, however, that the kinesin motor modulators ofthis invention are relatively charged molecules. Both charge andmolecule size tend to decrease cellular uptake and serum half-life.Consequently, in a preferred embodiment, it is desirable to package,complex, or otherwise combine the kinesin motor modulator with adelivery vehicle that preferably increases cellular uptake and/or serumhalf-life.

[0142] A wide variety of suitable vehicles are well known to those ofskill. Thus, for example, the kinesin motor modulator can be complexedwith, or encapsulated within, a charged lipid to form a net neutralcomposition. This will reduce clearance by the reticuloendothelialsystem and enhance cellular uptake.

[0143] In another embodiment, the kinesin motor modulators can beencapsulated within or complexed with microparticles which can berecognized and phagocytosed by a target cell thereby facilitating entryof the kinesin motor modulator into the cell. Other methods offacilitating entry include the use of fusion proteins, proteincomplexes, and masking charged sulfate groups with reversible chemicalmodification or counterions.

[0144] The size of particles and their mode of delivery determines theirbiological behavior. Strand et al. (1998) in Microspheres-BiomedicalApplications, A. Rembaum, ed., pp 193-227, CRC Press have described thefate of particles to be dependent on their size. Particles in the sizerange of a few nanometers (nm) to 100 nm enter the lymphatic capillariesfollowing interstitial injection, and phagocytosis may occur within thelymph nodes. After intravenous/intraarterial injection, particles lessthan about 2 microns will be rapidly cleared from the blood stream bythe reticuloendothelial system (RES), also known as the mononuclearphagocyte system (MPS). Particles larger than about 7 microns will,after intravenous injection, be trapped in the lung capillaries. Afterintraarterial injection, particles are trapped in the first capillarybed reached. Inhaled particles are trapped by the alveolar macrophages.It will also be appreciated that microparticles, and other deliveryvehicles can be targeted to specific cells and/or tissues (e.g., byconjugation with antibodies, or other cell or tissue specific ligands)or by the use of vehicles that have specific cell or tissue trophisms.

[0145] While the kinesin motor modulators of this invention aregenerally water soluble, some species are moderately insoluble. Thosecompounds that are water-insoluble or poorly water-are not well suitedto conventional administration (e.g., by intravenous injection or oraladministration). The parenteral administration of such pharmaceuticalscan be achieved by emulsification of oil or lipid solubilized compoundwith an aqueous liquid (such as normal saline) in the presence ofsurfactants or emulsion stabilizers to produce stable microemulsions.These emulsions may be injected intravenously, provided the componentsof the emulsion are pharmacologically inert. For example, U.S. Pat. No.4,073,943 describes the administration of water-insolublepharmacologically active agents dissolved in oils and emulsified withwater in the presence of surfactants such as egg phosphatides, pluronics(copolymers of polypropylene glycol and polyethylene glycol),polyglycerol oleate, etc. PCT International Publication No. WO85/00011describes pharmaceutical microdroplets of an anaesthetic coated with aphospholipid, such as dimyristoyl phosphatidylcholine, having suitabledimensions for intradermal or intravenous injection.

[0146] Additionally, protein microspheres have been utilized as carriersof pharmacological or diagnostic agents. Microspheres of albumin havebeen prepared by either heat denaturation or chemical crosslinking. Heatdenatured microspheres are produced from an emulsified mixture (e.g.,albumin, the agent to be incorporated, and a suitable oil) attemperatures between 100° C. and 150° C. The microspheres are thenwashed with a suitable solvent and stored. Leucuta et al. (1988)International Journal of Pharmaceutics 41: 213-217, describe the methodof preparation of heat denatured microspheres.

[0147] For certain of the therapeutic uses of the subject kinesin motormodulators, particularly cutaneous uses such as for the control ofkeratinocyte proliferation, direct (e.g., topical or injected)administration of the kinesin motor modulator will be appropriate.Accordingly, the subject kinesin motor modulator, alone or incombination with a delivery vehicle, may be conveniently formulated foradministration with a biologically acceptable medium, such as water,buffered saline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) or suitable mixtures thereof. Inpreferred embodiments, the kinesin motor modulator is dispersed in lipidformulations, such as miscelles, which closely resemble the lipidcomposition of natural cell membranes to which the kinesin motormodulator is to be delivered.

[0148] As indicated above, the kinesin motor modulators are preferablycombined with a pharmaceutically acceptable carrier for in vivoadministration. Pharmaceutically acceptable carriers (excipients) cancontain a physiologically acceptable compound that acts, for example, tosolubilize the composition, and/or to stabilize the composition, and/orto increase or decrease the absorption of the agent. Physiologicallyacceptable compounds can include, for example, carbohydrates, such asglucose, sucrose, or dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low and/or high molecular weightproteins, compositions that reduce the clearance or hydrolysis of thekinesin motor modulator(s), or excipients or other stabilizers and/orbuffers. Other physiologically acceptable compounds include wettingagents, emulsifying agents, dispersing agents or preservatives which areparticularly useful for preventing the growth or action ofmicroorganisms.

[0149] For those kinesin motor modulators that are lipid soluble the useof solubilizers and/or emulsifiers is often desired to produce aqueouskinesin motor modulator solutions or emulsions. Such solubilizers andemulsifiers are well known to those of skill in the art.

[0150] For example, lower alkyl alcohols having from 2 to 3 carbon atomsare useful as diluents or solvents for kinesin motor modulators in thepreparation of stabilized kinesin motor modulator compositions of theinvention. Particularly useful alcohols are selected from the groupconsisting of ethyl alcohol, n-propyl alcohol and mixtures thereof.These alcohols are useful generally in the proportions by weight ofabout 1 to about 25 percent, preferably about 3 to about 15 percent,more preferably about 4 to about 10 percent, and most preferably about 4to about 6 percent by weight, all based upon the weight of the kinesinmotor modulator. These alcohols are miscible in both water and many oilsand can, therefore, be utilized as solvents for most of the forms of thefat-soluble kinesin motor modulators. These alcohols also serve tocontrol the viscosity of the kinesin motor modulator composition and actas secondary emulsifiers. Additionally, the alcohols can act as freezedepressants maintaining the fluidity of the kinesin motor modulatorcomposition at lower temperatures.

[0151] The emulsifier system optionally utilized in the kinesin motormodulator compositions of this invention can be selected from thevarious ionic or nonionic emulsifiers. The emulsifiers used must beacceptable as additives for oral administration and/or for intravenousadministration and have no significant deleterious effect upon thekinesin motor modulator used therewith or upon the effectiveness of thelower alkyl alcohol utilized as a solvent or diluent.

[0152] The kinesin motor modulator based pharmacological compositionsare preferably sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like.

[0153] For preparing pharmaceutical compositions from the compounds ofthe present invention, pharmaceutically acceptable carriers can beeither solid or liquid. Solid form preparations include powders,tablets, pills, capsules, cachets, suppositories, and dispersiblegranules. A solid carrier can be one or more substances which may alsoact as diluents, flavoring agents, solubilizers, lubricants, suspendingagents, binders, preservatives, tablet disintegrating agents, or anencapsulating material.

[0154] In powders, the carrier is a finely divided solid which is in amixture with the finely divided active component. In, tablets, theactive component is mixed with the carrier having the necessary bindingcapacity in suitable proportions and compacted in the shape and sizedesired.

[0155] The powders and tablets preferably contain from five or ten toabout seventy percent of the active compound. Suitable carriers aremagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as carrier providing acapsule in which the active component, with or without carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid formssuitable for oral administration.

[0156] It is recognized that the kinesin motor modulators, whenadministered orally, must be protected from digestion. This is typicallyaccomplished either by complexing the kinesin motor modulator with acomposition to render it resistant to acidic and enzymatic hydrolysis orby packaging the kinesin motor modulator in an appropriately resistantcarrier such as a liposome. Means of protecting compounds from digestionare well known in the art (see, e.g., U.S. Pat. No. 5,391,377 describinglipid compositions for oral delivery of therapeutic agents)

[0157] For preparing suppositories, a low melting wax, such as a mixtureof fatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogenous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

[0158] Liquid form preparations include solutions, suspensions, andemulsions, for example, water or water propylene glycol solutions. Forexample, parenteral injection liquid preparations can be formulated insolutions in aqueous polyethylene glycol solution.

[0159] Aqueous solutions suitable for oral use can be prepared bydissolving the active component in water and adding suitable colorants,flavors, stabilizing and thickening agents, as desired.

[0160] Aqueous suspensions suitable for oral use can be made bydispersing the finely divided active component in water with viscousmaterial, such as natural or synthetic gums, resins, methylcellulose,sodium carboxymethylcellulose, and other well known suspending agents.

[0161] Also included are solid form preparations which are intended tobe converted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

[0162] The concentration of kinesin motor modulators or other activeingredients in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe patient's needs.

[0163] C) Dosages.

[0164] Where the kinesin motor modulator is used in a therapeuticcontext, (e.g., in the treatment of a condition characterized bycellular hyperproliferation), a therapeutically effective quantity ofadocia-derived kinesin modulator is employed in treatment. Atherapeutically effective quantity or dosage refers to a dosage adequateto ameliorate symptoms or signs of the disease or to provide effectiveprophylaxis without producing unacceptable toxicity to the patient. Ingeneral, an effective amount of the compound is that which provideseither subjective relief of symptoms or an objectively identifiableimprovement as noted by the clinician or other qualified observer.

[0165] The dosage of compounds used in accordance with this inventionvaries depending on the compound and the condition being treated. Theage, weight, and clinical condition of the recipient patient; and theexperience and judgment of the clinician, practitioner, or veterinarianadministering the therapy are among the factors affecting the selecteddosage. Other factors include the route of administration the patient,the patient's medical history, the severity of the disease process, andthe potency of the particular compound.

[0166] Broadly, a dosing schedule is from about 2 mg to about 2000 mgtwo or three times a day. More typically, a dose is about 20 mg to about400 mg of compound given three times a day.

[0167] A dosage range for topical treatment is about 0.1% to about 10%(weight/volume) in a physiologically acceptable eye drop applied one tofive or even ten times a day.

[0168] It will be appreciated that such dosages are typically advisorialin nature and may be adjusted depending on the particular therapeuticcontext, patient tolerance, etc. Substantially higher dosages arepossible by any selected route, for example, topical administration.

[0169] Typically, the dosage is administered at least once a day until atherapeutic or prophylactic result is achieved. Preferably, the dosageis administered twice a day, but more or less frequent dosing can berecommended by the clinician. Once a therapeutic result is achieved, thedrug level can be modified for maintenance treatment. Under someconditions, the drug may be tapered or discontinued after the appearanceof a therapeutic result. Occasionally, side effects warrantdiscontinuation of therapy.

[0170] V. Adocia-Derived Kinesin Motor Modulator Kits.

[0171] In another embodiment, this invention provides kits for thepractice of the methods of this invention. The kits preferably includeone or more containers containing a adocia-derived kinesin motormodulator of this invention. The kit can optionally include apharmaceutically acceptable excipient and/or a delivery vehicle (e.g., aliposome). The kinesin modulator may be provided suspended in theexcipient and/or delivery vehicle or may be provided as a separatecomponent which can be later combined with the excipient and/or deliveryvehicle. The kit may optionally contain additional therapeutics to beco-administered with the kinesin motor modulator.

[0172] The kits may also optionally include appropriate systems (e.g.opaque containers) or stabilizers (e.g. antioxidants) to preventdegradation of the kinesin motor modulators by light or other adverseconditions.

[0173] The kits may optionally include instructional materialscontaining directions (i.e., protocols) providing for the use of aAdocia-derived kinesin motor modulator in the treatment of a disease ina mammal wherein the disease is characterized by cellularhyperproliferation. In particular the disease can include any one ormore of the disorders described herein.

[0174] While the instructional materials typically comprise written orprinted materials they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

EXAMPLES

[0175] The following examples are offered to illustrate, but not tolimit the present invention.

Example 1 Isolation of Adociasulfates

[0176] The sponge Adocia (Haliclona) sp. (Collection # 95-100) wascollected in Palau, Western Caroline Islands, and was quickly frozen.The frozen sponge (225 g) was diced and steeped in a mixture ofdichloromethane (300 mL) and methanol (1 L) for 24 h. The solids wereremoved by filtration and the solution was reduced in volume to 300 mLand extracted with dichloromethane (2×200 mL).

[0177] The aqueous phase was lyophilized to yield a pale yellow powder.The powder (1.0 g) was chromatographed twice on a reversed phase C18Sep-Pak, using a gradient of 30% MeOH in H₂₀ to 100% methanol (MeOH) aseluant, to obtain pure fractions containing adociasulfate-1 (FIG. 1a)and adociasulfate-2 (FIG. 1b) and a mixed fraction containingadociasulfates (FIGS. 1a, 1 b, and 1 c). The mixed fraction wasseparated by reversed phase HPLC using 1:1 MeOH—H₂O as eluant. Purefractions were combined to obtain adociasulfate-1 (13.5 mg),adociasulfate-2 (14.1 mg) and adociasulfate-3 (3.3 mg).

Example 2 Screening and Identification of Adocia-derived KinesinModulators

[0178] Motility Assay.

[0179] TI-γ (a kinesin superfamily member from the fungus Thermomyceslanuginosus) was adsorbed to a glass coverslip and supplemented with amixture of microtubules, 2 mM Mg-ATP, and sponge extracts in DMSO (5%final concentration). Motility was scored visually on a Zeiss Axioplanmicroscope sat up for DIC and fitted with an Argus 10 video processor(Hamamatsu).

[0180] Proteins.

[0181] All kinetic and binding measurements were performed on abacterially expressed Drosophila kinesin heavy chain fragment containingamino acids 5-351 of the wild type protein and a hexahistidine tag atthe C-terminus. Protein was purified from the soluble fraction of IPTGinduced bacterial cells by a single round of affinity chromatography onNi-NTA-agarose (Qiagen), concentrated by microfiltration, and frozen insmall aliquots in liquid nitrogen.

[0182] Steady State Kinetics.

[0183] Initial rate measurements were done at room temperature using amalachite green assay (Geladopoulos et al. (1991) Anal. Biochem., 192:112-116) modified to work in 96 well microtiter plates and scored on aplate reader at 650 run. ATP concentration dependence and basal ATPaserate were determined by a coupled enzymatic assay with pyruvate kinaseand lactate dehydrogenase monitoring changes in absorbance at 340 nm.Phosphate standards (650 μM-7 μM) were included with each reading.

[0184] ATPase Assay (ADP Release).

[0185] The percent of ADP released from the enzyme was determined by themethods of Hackney (see, e.g., Hackney (1994) J. Biol. Chem., 2690:16508-16511). Briefly, 80 μM kinesin was preincubated with α-³²PATP atroom temperature for 15 min and than stored on ice. 1 μl aliquots ofthat mixture were diluted into 100 μl of “chase mix” containing 0.5mg/ml pyruvate kinase, 2 mM phosphoenalpyruvate, and varyingconcentrations of adociasulfate. At different time points 5 μl aliquotsof the chase mix were quenched in 100 μl 1 M HCV/1 mM ATP/1 mM ADP. Theamount of ADP that became accessible to pyruvate kinase and wasconverted to ATP was determined by a thin layer chromatography onPEI-cellulose followed by phosphoimager quantitation.

[0186] In Vivo Assays.

[0187] The effects of adociasulfate (FIG. 1a) injection in the earlypre-cellular blastoderm embryo of Drosophila melanogaster wereevaluated. Embryos were collected every 20 minutes, dechorionated, andprepared for injection (Santamaria (1986) paged 159-174 in Drosophila, APractical Approach, D. B. Roberts, Eds., IRL Press, Oxford). The embryoswere desiccated for 7 minutes and pressure injected with either theadociasulfate solution in injection buffer (5 mM KCl, 100 mM sodiumphosphate pH 7.5), or with buffer alone as control The injected volumewas less than 10% of the total embryonic volume. Sets of 20 embryos wereinjected in each batch and at least three such batches were injected foreach different concentration of adociasulfate and control. The embryoswere then allowed to develop for 20-30 minutes at room temperatureinside a moist chamber and were subsequently fixed, devitelinized andimmunostained for tubulin, and counter stained with 0.01 mg/ml DAPI(Ashbumer, Drosophila, a Laboratory Manual, Cold Spring HarborLaboratory Press, New York. (1989)).

[0188] Results

[0189] Extracts from 268 marine sponges were initially tested for theirability to disrupt normal behavior of microtubules in a gliding motilityassay. This screening method allowed immediate distinction betweensubstances that affected microtubule movement and those that causedmicrotubule depolymerization or breakage. Active extracts from theinitial screening were then tested for inhibition of themicrotubule-stimulated kinesin ATPase.

[0190] The most promising candidates were extracts from the spongeAdocia sp. In the motility assay, these extracts disrupted microtubuleattachment to the kinesin-coated surface, and totally abolishedmovement. The microtubule stimulated ATPase of kinesin was alsocompletely inhibited.

[0191] Three active compounds in the extract were identified andisolated (see FIGS. 1a, 1 b, and 1 c). These specific compounds arereferred to herein as adociasulfates while the generic compounds arereferred to as Adocia compounds or Adocia kinesin modulators (e.g.,inhibitors). The structure of the Adocia compounds or adociasulfatesdoes not resemble that of nucleotide triphosphates. This indicates thatthe Adocia structures are different from known kinesin modulators. Inaddition, it is believed that the activity spectrum of the Adociacompounds is narrower than that of nucleotide triphosphates or analoguesthereof.

[0192] To further investigate specificity, the adociasulfate of FIG. 1awas tested on a variety of ATPases using the ATPase activity assaydescribed above. Of those tested, the only enzymes substantiallyinhibited by adociasulfate are members of kinesin superfamily (Table 2).TABLE 2 Concentrations of adociasulfate causing 50% inhibition ofenzymatic activity. enzyme C50 rabbit kidney ATPase >136 μM*Apyrase >136 μM* Myosin II (EDTA)^(A)    75 μM CENP-E    10 μM^(D)K5-351^(B)    2 μM^(E) K411^(C)    2 μM^(E) TI-γ    2 μM^(E) ncd —myosin — pyruvate kinase —

[0193] The behavior observed in the motility assay indicated thatadociasulfates interfere with microtubule binding to the motor. This wastested by performing a kinesin-microtubule co-sedimentation assay in thepresence of a nonhydrolysable ATP analog, AMP-PNP, with or withoutadociasulfate (FIG. 3). Addition of adociasulfate abolished binding ofkinesin to microtubules under these conditions.

[0194] Consideration of the kinesin mechanochemlcal cycle suggests thatthe effect on microtubule binding could be induced either by looking thekinesin in a weakly-binding state resembling the kinesin-ADPintermediate by adociasulfate binding in the nucleotide pocket, or bydirect interference with the microtubule-binding site. Steady statekinetic measurements demonstrated that the adociasulfate-inducedinhibition is competitive with microtubules, and could be totallyreversed by high microtubule concentrations (FIG. 4a).

[0195] In contrast, varying the ATP concentration had no effect on theoverall shape of the kinetic curves. V_(max) was progressively lower athigher adociasulfate concentrations (FIG. 4b). An additional argumentagainst adociasulfate binding at the nucleotide pocket comes from thelack of an inhibitory effect on the basal, non microtubule-stimulatedrate of the kinesin ATPases. If adociasulfate interfered with nucleotidebinding, or locked the enzyme in a particular nucleotide-bound state,ATP turnover in the absence of microtubule should be decreased. However,concentrations of up to 136 μM adociasulfate (the highest tested) didnot inhibit the basal ATPase rate.

[0196] Microtubule binding to kinesin induced 1,000-fold stimulation ofthe basal ATPase rate, owing primarily to accelerated ADP release. Iswas tested whether adociasulfate binding to kinesin could mimic theeffect of the microtubule by examining ADP release from kinesin in thepresence of varying concentrations of adociasulfate. Indeed, bursts ofADP release were observed and their magnitude correlated positively withthe concentration of adociasulfate (FIGS. 5a and 5 b). The adociasulfateconcentration at 50% of maximum burst is much higher than the K_(i)determined in steady state microtubule competition assays. Thisdiscrepancy may reflect different affinities for adociasulfate indifferent nucleotide states of kinesin. Steady state kineticmeasurements of K_(i) reflect the affinity of the most tightly boundstate of the entire cycle, which includes several kinesin-nucleotideintermediates (K-ATP, K-ADP-Pi, K-ADP etc.).

[0197] In contrast, the ADP release experiment with adociasulfateinvolved only one state, K-ADP. It is intriguing that this state alsohas the lowest affinity for the microtubule. It was initially surprisingthat adociasulfate did not stimulate the kinesin basal ATPase eventhough it induced ADP release. However, during steady state kineticmeasurements, each single headed kinesin molecule must undergo severalcycles of attachment-detachment to microtubule subunits. In contrast, ASpresumably remains bound through multiple enzymatic turnovers. Thephysiological equivalent of such a state would be a kinesin moleculepermanently attached to a single tubulin dimer, a state for which nokinetic data exist. However, if the adociasulfate binding to kinesinresembles microtubule binding, the initial association event shouldresult in a burst of ADP release as observed.

Example 3 In Vivo Effects of Adociasulfates

[0198] The in vivo effects of adociasulfate were also investigated.Preliminary experiments demonstrated that AS had no effect on HeLa cellsproliferation. However, because it is a fairly large molecule (MW 738)with two charged sulfate moieties, adociasulfate may have problemscrossing the cell membrane. This difficulty can be alleviated by directinjection of adociasulfate into a cell.

[0199] Drosophila embryos, a well characterized and sturdy system, werethus used for direct injection studies. In Drosophila embryos the first13 rounds of cell division take place in a syncitium during the firsttwo hours after fertilization. The first seven of these divisions aresynchronized and occur at the center of the egg yolk. During theremaining divisions, most nucleic move to the surface of the egg andcontinue to divide for the next three times in partially open cell buds(see, Campos-Ortega et al. (1985) The embryonic development ofDrosophila melanogaster, Springer Verlag). Thus, any drug injected in tothe Drosophila will have access to a large number of mitotic nuclei atthe surface of the egg during the last three divisions.

[0200] Three different concentrations of adociasulfate were injected (1mM, 0.1 mM, and 0.05 mM) in combination with 1 mg/mltetramethylrhodamine (TMR)-tubulin. Injection of up to 0.1 mMadociasulfate arrested all nuclear divisions immediately at the point ofinjection. Injection of 0.05 mM adociasulfate caused less severephenotypes that allowed observation of more distinct abnormalities.Spindles and microtubule asters without chromosomes were found at thesite of injection. Mats of unattached microtubules and chromosomesapparently detached from the spindle were also observed. These effectscould be accounted for by loss of function of various members of thekinesin superfamily.

[0201] These results demonstrate that adociasulfate specificallymodulats kinesin activity by interfering with microtubule binding. Thismechanism is unlike that of any know kinesin (or other motor) inhibitor.Without being bound to a theory it is believed that adociasulfatemodulats binding by emulating tubulin binding to a portion of themicrotubule binding site of kinesin. In addition, adociasulfate is apotent toxin, which, when delivered intracellularly, may ablate several,if not all, aspects of kinesin-superfamily mediated transport.

[0202] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference.

What is claimed is:
 1. A compound having a formula

wherein R¹ and R² are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R¹ and R² isnot H, or R¹ and R² are combined to form a single divalent terpenemoiety selected from the group consisting of hemiterpenes, terpenemonomers and terpene oligomers; X¹ and X² are the same or different andare anionic derivatives of an organic group, an inorganic group or agroup which is a combination of organic and inorganic groups; Y^(+n) isan organic or inorganic cation; m is either 1 or 2; and n is either 1 or2.
 2. A compound according to claim 1, wherein said terpene oligomersare members selected from the group consisting of sesquiterpenes,diterpenes, sesterterpenes, triterpenes, tetraterpenes and meroterpenes.3. A compound according to claim 1, wherein X¹ and X² are independentlyanions derived from members selected from the group consisting of formicacid, acetic acid, glycolic acid, phosphoric acid, phosphonic acid,phosphinic acid, boronic acid, sulfuric acid, and arsonic acid; andY^(+n) is a member selected from the group consisting of inorganiccations and tetralkylammonium salts.
 4. A compound according to claim 3,wherein R¹ is hydrogen; R² is a member selected from the groupconsisting of diterpenes, triterpenes and meroterpenes; and X¹ and X²are —OSO₃ ⁻; n is 1; and m is
 2. 5. A compound according to claim 1,wherein Y is a tetraalkylammonium salt according to the formula(R⁴)₃N—(CH₂)_(t)—R³—(CH₂)_(s)—N(R⁴)₃ wherein R³ is a member selectedfrom the group consisting of C₁-C₁₀ aryl, substituted aryl, alkyl andsubstituted alkyl groups; R⁴ is lower alkyl or substituted lower alkyl;m is 1; n is 2; s is an integer between 1 and 5; and t is an integerbetween 1 and
 5. 6. A compound according to claim 1 having a formula

wherein R⁵ and R⁶ are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R⁵ and R⁶ isnot H, or R⁵ and R⁶ are combined to form a single divalent terpenemoiety selected from the group consisting of hemiterpenes, terpenemonomers and terpene oligomers.
 7. A compound according to claim 6,wherein said terpene oligomers are members selected from the groupconsisting of sesquiterpenes, diterpenes, sesterterpenes, triterpenes,tetraterpenes and meroterpenes.
 8. A compound according to claim 1having the formula


9. A compound according to claim 1 having the formula:


10. A compound according to claim 1 having the formula


11. A composition for in vivo modulation of kinesin motor activity, saidcomposition comprising: a pharmaceutically acceptable excipient; and acompound having the formula

wherein R¹ and R² are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R¹ and R² isnot H, or R¹ and R² are combined to form a single divalent terpenemoiety selected from the group consisting of hemiterpenes, terpenemonomers and terpene oligomers; X¹ and X² are the same or different andare anionic derivatives of an organic group, an inorganic group or agroup which is a combination of organic and inorganic groups; Y^(+n) isan organic or inorganic cation; m is either 1 or 2; and n is either 1 or2.
 12. The composition according to claim 11, wherein said terpeneoligomers are members selected from the group consisting ofsesquiterpenes, diterpenes, sesterterpenes, triterpenes, tetraterpenesand meroterpenes
 13. The composition according to claim 11, wherein X¹and X² are independently anions derived from members selected from thegroup consisting of formic acid, acetic acid, glycolic acid, phosphoricacid, phosphonic acid, phosphinic acid, boronic acid, sulfuric acid, andarsonic acid; and Y^(+n) is a member selected from the group consistingof inorganic cations and tetralkylammonium salts.
 14. The compositionaccording to claim 13, wherein X¹ and X² are the same.
 15. Thecomposition according to claim 13, wherein R¹ is hydrogen; R² is amember selected from the group consisting of diterpenes, triterpenes andmeroterpenes; and X¹ and X² are —OSO₃ ⁻; n is 1; and m is 2
 16. Thecomposition according to claim 11, wherein Y is a tetraalkylammoniumsalt according to the formula (R⁴)₃N—(CH₂)_(t)—R³—(CH₂)_(s)—N(R⁴)₃wherein R³ is a member selected from the group consisting of C₁-C₁₀aryl, substituted aryl, alkyl and substituted alkyl groups; R⁴ is loweralkyl or substituted lower alkyl; m is 1; n is 2; s is an integerbetween 1 and 5; and t is an integer between 1 and
 5. 17. Thecomposition according to claim 11, having a formula:

wherein R⁵ and R⁶ are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R⁵ and R⁶ isnot H, or R⁵ and R⁶ are combined to form a single divalent terpenemoiety selected from the group consisting of hemiterpenes, terpenemonomers and terpene oligomers.
 18. The composition according to claim17, wherein said terpene oligomers are members selected from the groupconsisting of sesquiterpenes, diterpenes, sesterterpenes, triterpenes,tetraterpenes and meroterpenes.
 19. The composition according to claim11, having the formula


20. The composition according to claim 11, having the formula


21. The composition according to claim 11 having the formula


22. A method for modulating kinesin motor activity in a cell, saidmethod comprising the step of: (i) contacting s aid cell with a compoundhaving the formula

wherein R¹ and R² are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R¹ and R² isnot H, or R¹ and R² are combined to form a single divalent terpenemoiety selected from the group consisting of hemiterpenes, terpenemonomers and terpene oligomers; X¹ and X² are the same or different andare anionic derivatives of an organic group, an inorganic group or agroup which is a combination of organic and inorganic groups; Y^(+n) isan organic or inorganic cation; m is either 1 or 2; and n is either 1 or2.
 23. The method of claim 22, wherein said terpene oligomers aremembers selected from the group consisting of sesquiterpenes,diterpenes, sesterterpenes, triterpenes, tetraterpenes and meroterpenes.24. The method of claim 22, wherein X¹ and X² are independently anionsderived from members selected from the group consisting of formic acid,acetic acid, glycolic acid, phosphoric acid, phosphonic acid, phosphinicacid, boronic acid, sulfuric acid, and arsonic acid; and Y^(+n) is amember selected from the group consisting of inorganic cations andtetralkylammonium salts.
 25. The method of claim 24, wherein X¹ and X²are the same.
 26. The method of claim 24, wherein R¹ is hydrogen; R² isa member selected from the group consisting of diterpenes, triterpenesand meroterpenes; and X¹ and X² are —OSO₃′; n is 1; and m is
 2. 27. Themethod of claim 22, wherein Y is a tetraalkylammonium salt according tothe formula (R⁴)₃N—(CH₂)_(t)—R³—(CH₂)_(s)—N(R⁴)₃ wherein R³ is a memberselected from the group consisting of C₁-C₁₀ aryl, substituted aryl,alkyl and substituted alkyl groups; R⁴ is lower alkyl or substitutedlower alkyl; m is 1; n is 2; s is an integer between 1 and 5; and t isan integer between 1 and
 5. 28. The method of claim 22, wherein saidcompound has the formula

wherein R⁵ and R⁶ are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R⁵ and R⁶ isnot H, or R⁵ and R⁶ are combined to form a single divalent terpenemoiety selected from the group consisting of hemiterpenes, terpenemonomers and terpene oligomers.
 29. The method of claim 28, wherein saidterpene oligomers are members selected from the group consisting ofsesquiterpenes, diterpenes, sesterterpenes, triterpenes, tetraterpenesand meroterpenes
 30. The method of claim 22, wherein said compound hasthe formula


31. The method of claim 22, wherein said compound has the formula


32. The method of claim 22, wherein said compound has the formula


33. The method of claim 22, wherein said cell is an animal cell.
 34. Themethod of claim 22, wherein said cell is a neuron.
 35. The method ofclaim 22, wherein said cell is a plant cell.
 36. The method of claim 22,wherein said cell is a fungal cell.
 37. The method of claim 22, whereinsaid cell is a tumor cell.
 38. A method of assaying a test compound forkinesin modulatory activity, said method comprising the steps of: (i)contacting a microtubule and a kinesin motor with said test compoundhaving the formula

wherein R¹ and R² are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R¹ and R² isnot H, or R¹ and R² are combined to form a single divalent terpenemoiety selected from the group consisting of hemiterpenes, terpenemonomers and terpene oligomers; X¹ and X² are the same or different andare anionic derivatives of an organic group, an inorganic group or agroup which is a combination of organic and inorganic groups; Y^(+n) isan organic or inorganic cation; m is either 1 or 2; and n is either 1 or2; and (ii) detecting a change in kinesin motor activity resulting fromsaid contacting.
 39. The method of claim 22, wherein said terpeneoligomers are members selected from the group consisting ofsesquiterpenes, diterpenes, sesterterpenes, triterpenes, tetraterpenesand meroterpenes.
 40. The method of claim 38, wherein X¹ and X² areindependently anions derived from members selected from the groupconsisting of formic acid, acetic acid, glycolic acid, phosphoric acid,phosphonic acid, phosphinic acid, boronic acid, sulfuric acid, andarsonic acid; and Y^(+n) is a member selected from the group consistingof inorganic cations and tetralkylammonium salts.
 41. The method ofclaim 40, wherein X¹ and X² are the same.
 42. The method of claim 40,wherein R¹ is hydrogen; R² is a member selected from the groupconsisting of diterpenes, triterpenes and meroterpenes; and X¹ and X²are —OSO₃—; n is 1; and m is
 2. 43. The method of claim 38, wherein Y isa tetraalkylammonium salt according to the formula(R⁴)₃N—(CH₂)_(t)—(CH₂)_(s)—N(R⁴)₃ wherein R³ is a member selected fromthe group consisting of C₁-C₁₀ aryl, substituted aryl, alkyl andsubstituted alkyl groups; R⁴ is lower alkyl or substituted lower alkyl;m is 1; n is 2; s is an integer between 1 and 5; and t is an integerbetween 1 and
 5. 44. The method of claim 38, wherein said compound hasthe formula

wherein R⁵ and R⁶ are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R⁵ and R⁶ isnot H, or R⁵ and R are combined to form a single divalent terpene moietyselected from the group consisting of hemiterpenes, terpene monomers andterpene oligomers.
 45. The method of claim 44, wherein said terpeneoligomers are members selected from the group consisting ofsesquiterpenes, diterpenes, sesterterpenes, triterpenes, tetraterpenesand meroterpenes.
 46. The method of claim 38, wherein said detectingcomprises a motility assay.
 47. The method of claim 38, wherein saiddetecting comprises a microtubule kinesin binding assay.
 48. The methodof claim 38, wherein said detecting comprises an ATPase assay.
 49. Themethod of claim 38, wherein said detecting comprises an anti-mitoticassay in situ, in vitro, or in vivo.
 50. The method of claim 38, whereinsaid change is with reference to a control assay lacking said testcompound.
 51. A kit for modulating kinesin motor activity, said kitcomprising a container containing compound having a formula

wherein R¹ and R² are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R¹ and R² isnot H, or R¹ and R² are combined to form a single divalent terpenemoiety selected from the group consisting of hemiterpenes, terpenemonomers and terpene oligomers; X¹ and X² are the same or different andare anionic derivatives of an organic group, an inorganic group or agroup which is a combination of organic and inorganic groups; Y^(+n) isan organic or inorganic cation; m is either 1 or 2; and n is either 1 or2.
 52. The kit according to claim 51, wherein said terpene oligomers aremembers selected from the group consisting of sesquiterpenes,diterpenes, sesterterpenes, triterpenes, tetraterpenes and meroterpenes.53. The kit according to claim 51, wherein X¹ and X² are independentlyanions derived from a members selected from the group consisting offormic acid, acetic acid, glycolic acid, phosphoric acid, phosphonicacid, phosphinic acid, boronic acid, sulfuric acid, and arsonic acid;and Y^(+n) is a member selected from the group consisting of inorganiccations and tetralkylammonium salts.
 54. The kit according to claim 53,wherein X¹ and X² are the same.
 55. The kit according to claim 53,wherein R¹ is hydrogen; R² is a member selected from the groupconsisting of diterpenes, triterpenes and meroterpenes; and X is —OSO₃⁻; n is 1; and m is
 2. 56. The kit according to claim 51, wherein Y is atetraalkylammonium salt according to the formula(R⁴)₃N—(CH₂)_(t)—R³—(CH₂)_(s)—N(R⁴)₃ wherein R³ is a member selectedfrom the group consisting of C₁-C₁₀ aryl, substituted aryl, alkyl andsubstituted alkyl groups; R⁴ is lower alkyl or substituted lower alkyl;m is 1; n is 2; s is an integer between 1 and 5; and t is an integerbetween 1 and
 5. 57. The kit according to claim 51 having a formula

wherein R⁵ and R 6 are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R⁵ and R⁶ isnot H, or R⁵ and R⁶ are combined to form a single divalent terpenemoiety selected from the group consisting of hemiterpenes, terpenemonomers and terpene oligomers.
 58. The kit according to claim 57,wherein said terpene oligomers are members selected from the groupconsisting of sesquiterpenes, diterpenes, sesterterpenes, triterpenes,tetraterpenes and meroterpenes.
 59. The kit according to claim 51 havingthe formula


60. The kit according to claim 51 having the formula


61. The kit according to claim 51 having the formula


62. A method of identifying a compound that specifically modulates akinesin motor at a microtubule binding site, said method comprising thesteps of: (i) assaying for competitive inhibition of said motor by saidmolecule at a kinesin ATPase site; (ii) assaying for competitiveinhibition of said motor by said molecule at a microtubule binding site;(iii) identifying said small molecule as a kinesin modulator specific toa microtubule binding site when said small molecule is a competitivemodulator at said microtubule binding site, but not at said ATPase site.63. The method of claim 62, wherein said assaying comprises detectingATPase activity of said kinesin motor.
 64. The method of claim 62,wherein compound is a polypeptide.
 65. The method of claim 62, whereincompound is a nucleic acid.
 66. The method of claim 62, wherein compoundis an antibody.
 67. The method of claim 62, wherein compound is a smallorganic molecule.
 68. The method of claim 62, wherein compound is aninorganic molecule.
 69. A method of modulating kinesin motor activity,said method comprising contacting said kinesin motor with a smallorganic molecule that competitively inhibits said kinesin motor at amicrotubule binding site.
 70. The method of claim 69, wherein said smallorganic molecule is identified according to the method of claim
 62. 71.A method of modulating kinesin motor activity, said method comprisingcontacting said kinesin motor with a small organic molecule thatcompetitively inhibits said kinesin motor at a microtubule binding site.72. The method of claim 71, wherein said small organic molecule isidentified according to the method of claim
 62. 73. A method ofidentifying an agent that modulates the kinesin inhibitory activity ofan Adocia kinesin inhibitor, said method comprising: (i) contacting amicrotubule, a kinesin motor, and an Adocia kinesin inhibitor with acandidate agent; and (ii) detecting a change in the kinesin inhibitoryactivity of the Adocia kinesin inhibitor resulting from said contacting,wherein a change indicates the identification of an agent that modulatesthe kinesin inhibitory activity of the Adocia kinesin inhibitor.
 74. Amethod of identifying an agent that interferes with the binding of anAdocia kinesin inhibitor with a kinesin, said method comprising: (i)contacting a kinesin and an Adocia kinesin inhibitor with a candidateagent; and (ii) detecting a decrease in the binding of the Adociakinesin inhibitor with the kinesin resulting from said contacting,wherein a decrease indicates the identification of an agent thatinterferes with the binding of the Adocia kinesin inhibitor and thekinesin.
 75. A complex comprising an Adocia kinesin inhibitor and akinesin.
 76. A method of modulating cellular growth in an organism, saidmethod comprising administering to said organism a compositioncomprising a pharmaceutically acceptable carrier the compound of claim 1in a quantity sufficient to alter said cellular growth in an organism.77. The method of claim 76, wherein said organism is an animal.
 78. Themethod of claim 76, wherein said organism is an plant.